1. Field of the Invention
The ability to transfer exogenous genetic material into higher plants promises to provide enhanced opportunities for agricultural scientists to increase food production in the coming decades. At present, the primary focus of those interested in the genetic manipulation of higher plants has been in two areas: use of the Ti plasmid of Agrobacterium tumefaciens and use of vectors based on the Caulimoviruses. While these systems offer the potential to integrate exogenous DNA into the plant's genome, they each suffer from many drawbacks including plant host range, efficiency of transformation, lengthy manipulative procedures, and the like. It would thus be desirable to have an improved system for the genetic manipulation of plants.
Direct microinjection of DNA as practiced in animal cells has many advantages, including simplicity and very high transformation rates. Despite these advantages, the utilization of direct microinjection of DNA into plant cells has found only limited use. Direct microinjection of plant cells is complicated by the presence of a rigid cell wall not found in animal cells. While protoplasts lacking the cell wall can be formed, the microinjection of plant cell protoplasts is made difficult by their extreme fragility. Successful microinjection has been achieved by immobilizing plant protoplasts on a solid substrate. Such immobilization, however, prevents easy separation of injected and non-injected cells, and the small percentage of injected cells can only be followed for a few days. A strong selectable marker would be required to allow identification and recovery of transformed cells, and the use of such markers may result in the loss of a significant portion of the transformed material.
Thus, it would be desirable to provide a method for the direct microinjection of DNA and other macromolecules into plant protoplasts, which method provides a high rate of uptake of the injected DNA into specified compartments of the plant cell with high viability of the injected protoplasts.
2. Description of the Prior Art
Steinbiss and Stabel (1983) Protoplasma 116:223-227 teach the microinjection of macromolecules into plant protoplasts where the protoplasts are first attached to microscope cover slips using polylysine. Although functional, the attachment with polylysine reduces the protoplast viability and microinjection into the nucleus occurs infrequently. Griesbach (1983) P1. Mol. Biol. Rep. 4:32-37 discloses the microinjection of chromosomes into higher plant protoplasts that are either free-floating or suspended in agar. Injection into the nucleus was not obtainable and protoplast viability was severely reduced. The microinjection of DNA into mouse eggs using a holding pipette and a microinjection pipette is described in Lin (1966) Science 151:333-337. See also, Wagner et al. (1981) Proc. Natl. Acad. Sci. USA 78:6376-6380; Brinster et al. (1981) Cell 27:223-231; and Gordon and Ruddle (1981) Science 214:1244-1246, where the successful transfer and expression of genetic material into the genomes of newborn mice by microinjection is described. See also, Lawrence and Davies, Plant Cell Reports (1985) 4:33-35, who describe a protoplast microinjection technique.